Overexpression of AcEXPA23 Promotes Lateral Root Development in Kiwifruit

Kiwifruit is loved by consumers for its unique taste and rich vitamin C content. Kiwifruit are very sensitive to adverse soil environments owing to fleshy and shallow roots, which limits the uptake of water and nutrients into the root system, resulting in low yield and poor fruit quality. Lateral roots are the key organs for plants to absorb water and nutrients. Improving water and fertilizer use efficiency by promoting lateral root development is a feasible method to improve yield and quality. Expansin proteins plays a major role in lateral root growth; hence, it is important to identify expansin protein family members, screen key genes, and explore gene function in root development. In this study, 41 expansin genes were identified based on the genome of kiwifruit (‘Hongyang’, Actinidia chinensis). By clustering with the Arabidopsis thaliana expansin protein family, the 41 AcExpansin proteins were divided into four subfamilies. The AcExpansin protein family was further analysed by bioinformatics methods and was shown to be evolutionarily diverse and conserved at the DNA and protein levels. Based on previous transcriptome data and quantitative real-time PCR assays, we screened the candidate gene AcEXPA23. Overexpression of AcEXPA23 in kiwifruit increased the number of kiwifruit lateral roots.


Introduction
Actinidia L., belonging to the family Actinidiaceae, comprises a total of 75 taxa, including 54 species and 21 varieties [1]. Cultivars in production are mainly derived from the original variety of Actinidia chinensis Planch. var. chinensis, A. chinensis Planch. var. deliciosa (A. Chev.), and Actinidia arguta [2]. Kiwifruit is known as the "king of fruits" because of its unique taste and rich vitamin C content and is popular among consumers [2][3][4]. As of 2019, the kiwifruit planting area in China reached 250,000 ha, far exceeding the sum of the planting areas of other countries in the world and increasing annually [5]. Although the kiwifruit harvested area and output of China rank first in the world, the output per unit area ranks only 20th in the world, with clear import and export trade deficits [5]. Fruit yield and quality are closely related to the efficiency of water and nutrient uptake by plant roots. The root system of kiwifruit is composed of fleshy roots, is mainly distributed in the upper layer of the soil, and is easily affected by the surrounding soil environment [1]. To a certain extent, this limits the moisture and nutrient absorption efficiency of kiwifruit roots, resulting in low fruit production and poor quality [6].
Our previous study showed that exogenous brassinosteroid (BR) treatment in kiwifruit did significantly promote lateral root development [39]. For in-depth research, we focused, herein, on the expansin gene, the most downstream regulatory gene in current lateral root research. We performed stable genetic transformation of the selected candidate gene AcEXPA23 in kiwifruit. Our results showed that overexpression of AcEXPA23 significantly promoted the increase in lateral roots in kiwifruit. This is important for kiwifruit to absorb more water and nutrients through the lateral roots to improve yield and quality.

Phylogenetic Tree Analysis of the AcExpansin Protein Family
We identified 41 expansin protein family members in kiwifruit. To further classify the expansin gene in kiwifruit, a phylogenetic tree was constructed using 41 AcExpansin protein sequences and with all 36 Arabidopsis expansin protein sequences as references ( Figure 1, Table S1). The results showed that the AcExpansin protein family was divided into four subfamilies: AcEXPA, AcEXPB, AcEXLA, and AcEXLB. According to the classification results, the identified AcExpansin protein family members were renamed ( Table 1). The subfamily AcEXPA contained 28 genes (AcEXPA1-AcEXPA28), subfamily AcEXPB contained 6 genes (AcEXPB1-AcEXPB6), subfamily AcEXLA had 3 genes (AcEXLA1-AcEXLA3), and subfamily AcEXLB had 4 genes (AcEXLB1-AcEXLB4).

Physicochemical Properties of AcExpansin Protein Family
We further analysed the physicochemical properties of the 41 AcExpansins. As shown in Table 1, the length of the AcExpansin proteins ranged from 193 to 783 aa. The molecular weight and theoretical pI of the identified AcExpansin proteins ranged from 20.73 kDa to 86.02 kDa and 4.73 to 10.16, respectively. The average isoelectric point was 8.37 and 80% of the expansin proteins had isoelectric points greater than 8.00, indicating that most of the expansin proteins were alkaline. The expansin protein instability index was 22.97-46.03. The lipid solubility index of expansin proteins was 57.67-83.11, with an average value of 70.66. Among them, the lipid solubility index of five AcExpansin proteins exceeded 80.00, indicating that they belonged to the class of thermophilic proteins. The total average hydrophobicity of the 41 AcExpansin genes was −0.441-0.131, indicating they belonged to amphiprotic proteins with comparable hydrophobicity (>0 for hydrophobicity, <0 for hydrophilicity, and ±0.5 for amphiprotic proteins).

AcExpansin Protein Family Chromosomal Location
The kiwifruit V3 genome contains 29 chromosomes. To determine the genomic distribution, physical location analysis of 41 expansin genes was performed using the online tool MG2C. Our results showed that 38 AcExpansin genes were unevenly distributed on 22 chromosomes, whereas 3 members of the AcExpansin protein family were attributed to chromosomes that were undetermined ( Figure 2). Among the 38 AcExpansin genes, chromosome 3 contained 4 genes; chromosomes 9, 22, and 25 contained 3 genes each; chromosomes 1, 4, 8, 12, 19, 21, and 23 contained 2 genes each; and the other chromosomes contained 1 gene each.

Analysis of Conserved Domains and Gene Structure of the AcExpansin Protein Family
In total, eight conserved motifs (named motif 1-8) in AcExpansin proteins were identified using the TBtools software (v.1098696) ( Figure 3A). Figure 3 shows that genes from the same subfamily have similar motifs, indicating structural similarities between genes in the same group. Except for motif 8, the other motifs were widely distributed in the AcEXPA subfamily. Motif 2 was only present in the AcEXPA subfamily. Motif 8 was unique to the AcEXPB, AcEXLA, and AcEXLB subfamilies.  The gene structure of the 41 AcExpansin genes was analysed using TBtools and genomic DNA sequences ( Figure 3B). Most of the AcEXPA genes included three exons. The number of exons in AcEXPB genes was either three or four. The exon number of AcEXLB genes was either four or five. The exon numbers of the three AcEXLA genes were very different. AcEXLA1, AcEXLA2, and AcEXLA3 contained 11, 5, and 13 exons, respectively.

Screening of Candidate Expansin gene under BR and Brassinazole Treatment
Based on our previous transcriptome sequencing data (Submission ID: SUB9537634, BioProject ID: PRJNA726005), a heatmap was constructed to analyse the expression patterns of the AcExpansin protein family members with BR and brassinazole treatment ( Figure 5A). The results indicated that genes that were upregulated were mainly from the AcEXPA subfamily. Three AcExpansin genes (AcEXPA14, AcEXPA18, and AcEXPA23) were markedly induced by BR and were markedly reduced by brassinazole treatment. We further performed qRT-PCR assays for these three AcExpansin genes, among which AcEXPA23 had the highest expression level of 78-fold ( Figure 5B). Therefore, we selected AcEXPA23 as a candidate gene to further explore its function in lateral root development.

Subcellular Localisation of AcEXPA23
To determine the subcellular localisation of AcEXPA23, we fused the terminatorremoved CDS of AcEXPA23 to green fluorescent protein (GFP) under the control of the CaMV35S constitutive promoter. Using a polyethylene glycol-mediated procedure, 35S::AcEXPA23:GFP fusion proteins and 35S::GFP (Control) were transferred into Arabidopsis protoplasts. The results showed that control 35S::GFP was distributed throughout the whole cell, whereas 35S::AcEXPA23:GFP was detected in the cytoplasm of the Arabidopsis protoplasts ( Figure 6).

Transient Overexpression of AcEXPA23 in Kiwifruit
To investigate the role of AcEXPA23 in lateral root development, transient overexpression was performed in kiwifruit by hairy root infection technology. Laser confocal microscopy was used and a PCR assay was performed to identify positive seedlings ( Figure 7A,B). Seedlings in which the fluorescence signal of hairy roots could be observed and amplified from the eGFP sequence fragment were considered as positive plants. Compared with the control, plant overexpression AcEXPA23 increased by 2.2-times in the number of lateral roots in the hairy roots ( Figure 7C,D). The results indicated that AcEXPA23 plays an important role in the lateral root development of kiwifruit.

AcEXPA23 Overexpression in Kiwifruit Enhanced the Number of Lateral Roots
To confirm the roles of AcEXPA23 in kiwifruit, we obtained AcEXPA23-overexpressing plants of kiwifruit by transforming explants produced from leaf strips. Similar to transient overexpression, seedlings in which the fluorescence signal of hairy roots was observed and amplified from the eGFP sequence fragment were considered as positive plants ( Figure 8A,B). Finally, two overexpression lines were obtained. We observed that overexpression of AcEXPA23 significantly increased the number of lateral roots compared with that in wild-type kiwifruit ( Figure 8C). The number of lateral roots of both Line 1 and Line 2 was 2.45-times higher than that of the wild-type seedlings ( Figure 8D). The expression levels of AcEXPA23 in Line 1 and Line 2 were 17-and 63-times that in wild-type plants, respectively ( Figure 8E). Therefore, we conclude that AcEXPA23 plays an important role in regulating lateral root development in kiwifruit.

Discussion
The expansin gene is widespread in plants, ranging from algae to higher plants, indicating that the expansin protein family is important for the growth and development of plants [40]. Evolutionary analysis of expansins can provide valuable insights into the regulation of important agronomic traits in kiwifruit genetics and breeding. Our study identified 41 expansin genes in kiwifruit containing two conserved domains, doublepsi-beta barrel and pollen allergen domains, which are characteristic of other expansins identified to date [41]. In kiwifruit, out of the 41 expansins, the number of AcEXPA was dominant compared to the other expansin categories (AcEXPB; AcEXLA and AcEXLB), in line with findings of earlier studies in other species [42][43][44][45][46][47][48][49][50]. Segmental and tandem duplications have been reported to be two of the main causes of gene family expansion in plants [51]. This conclusion is further supported by the finding that most members of the AcExpansin subfamily undergo tandem and segmental duplications. Genes that undergo duplication have three evolutionary outcomes: maintaining the original conserved function, generating new functions, or forming pseudogenes [52]. AcEXPA1-AcEXPA16 are a pair of tandem duplicated genes located on chromosome 3, which are from the EXPA subfamily (Figures 1, 2 and 4). The expression of AcEXPA1 and AcEXPA16 was obviously different (Figure 5), indicating that they may have evolved into two genes with different functions. Furthermore, the motif compositions of these two genes were found to be consistent (Figure 3), suggesting that the difference in function may originate from the difference in cis-acting elements in the promoter region.
The expression of expansin genes is not only regulated by plant development processes but is also affected by plant hormones. For example, BR induces elongation of excised epicotyl segments and the levels of all EXPA transcripts increased significantly in Cicer arietinum [53]. BR promotes AtEXPA5 expression and positively affects root cell elongation [54]. The transcript level of EXP1 was upregulated in response to BR treatment in sweet potato [55]. Previously, with heat mapping and qRT-PCR analysis, we also found that BR treatment upregulates AcEXPA23 expression in kiwifruit roots ( Figure 5A) [39]. We analysed the interaction network of AcEXPA23 using STRING based on AcExpansin ortho-logues in Arabidopsis. This could help us to understand gene function and efficiency [56]. The similarity between AcEXPA23 and AtEXPA14 was 83.2%. Studies showed that overexpression of AtEXPA14 in Arabidopsis stimulated the formation of emerged lateral roots, whereas loss of function of AtEXPA14 reduced auxin-stimulated lateral root formation [18]. We speculate that the AcEXPA23 gene plays an important role in the regulation of lateral root development.
In this study, transformation experiments demonstrated that overexpression of AcEXPA23 could significantly enhance the number of lateral roots in kiwifruit (Figures 7 and 8), which was in line with findings of previous studies [18,[57][58][59][60]. A recent study on maize yield found that 48% of the yield gain was associated with a decadal climate trend, 39% with agronomic improvements, and only 13% with improvement in genetic yield potential [61]. These findings differed from those of most previous studies, which attributed a much greater weight to genetic yield potential improvement. The continuous activities of human beings have gradually intensified climate change, and it has become more and more important to improve the adaptability of plants [62][63][64]. The present study shows that overexpression of AcEXPA23 promotes the development of increased lateral roots in kiwifruit. Therefore, BR can be developed into a root regulation product as an improved agronomic measure to improve the root system of kiwifruit, improve the utilisation of water and nutrients, and improve yield and quality.
Expansins are cell-wall-loosening proteins. Most studies showed that expansins are localized in the cell wall, such as EXPB2 from rice [65], EXPB2 from Heterodera avenae [66], and EXPA4 from Chrysanthemum morifolium [67]. However, some expansins are also reportedly localised to the plasma membrane. For example, the subcellular localisation of epidermal cells in tobacco and onions revealed that EXPB7 was localised in the plasma membrane of Hordeum vulgare [68], EXPA1 was located in the plasma membrane [69], EXLA2 was also located in the plasma membrane of tobacco [70]. In the present study, the 35S::AcEXPA23:GFP fusion protein was transiently expressed in Arabidopsis protoplasts and the results showed that the protein was located in the cytoplasm (Figure 6), which may be related to the specific functions of expansins.

Identification of Kiwifruit Expansin Genes
We identified the expansin protein family genes in kiwifruit using the following four steps. First, the hidden Markov model (HMM) of the two characteristic domains of the expansin gene, pfam01357 (Pollen_allerg_1) and pfam03330 (DPBB_1), from the Pfam database (http://pfam.xfam.org/ (accessed on 4 January 2022)) was downloaded. The expansin protein family genes of kiwifruit were then identified using the Simple HMM Search function in the TBtools software [71]. Second, the Arabidopsis expansin protein family protein sequences were downloaded from the TAIR website (www.arabidopsis.org (accessed on 5 January 2022)). The expansin protein family genes of kiwifruit were identified using the Blast Compare Two Seqs function in the TBtools software [71]. Third, the intersection of the genes obtained in the above two steps was considered. Finally, according to the two characteristic domains of the expansin gene, pfam01357 (Pollen_allerg_1) and pfam03330 (DPBB_1), the final screening was performed on the website of InterPro (https://www.ebi.ac. uk/interpro/ (accessed on 6 January 2022)). In addition, the kiwifruit genome protein data used in the first and second identification processes were downloaded from the kiwifruit genome database (http://kiwifruitgenome.org (accessed on 4 January 2022)).

Analysis of AcExpansin Protein Family Characteristics
ClustalW in MEGA11 was used to align the relatedness of AcExpansin protein family gene sequences and a phylogenetic tree was constructed using the neighbour-joining method in MEGA11 (related parameter settings: bootstrap, 1000; model/method, pdistance; gaps/missing data treatment, partial deletion) [72]. Further adjustments and annotations to the evolutionary tree were performed using EVOLVIEW (http://www. evolgenius.info/evolview/#/ (accessed on 16 January 2022)). Chromosome localisation maps were generated online using MG2C (http://mg2c.iask.in/mg2c_v2.1/ (accessed on 21 January 2022)). Motif and gene structures were analysed and visualised using the TBtools software [71]. The physicochemical properties of the proteins were analysed using the online tool Expasy (https://www.expasy.org/ (accessed on 19 January 2022)). Intraspecies collinearity analysis was performed and visualised using the TBtools software [71].

Transient Expression Hairy Root
We performed the following steps for instantaneous conversion. The AcEXPA23 ORF was first cloned into the pART-CAM-EGFP vector under the control of the CaMV 35S promoter (Table S2). The recombinant vector was then transformed into K599 Agrobacterium. The K599 Agrobacterium containing the target gene was shaken to make the OD 600 value reach 0.5-0.8 and the volume of the bacterial solution was approximately 10 mL. The bacterial solution was then centrifuged twice at 8000 rpm for 10 min each and then resuspended in MES buffer (10 mmol/L MES-KOH, pH = 5.2, 10 mmol/L MgCl 2 and 10 µmol/L acetosyringone). The wild-type kiwifruit (rooted tissue culture seedlings) in good growth condition was selected, and 100-150 µL of bacterial liquid was injected into the young stem with a 0.5 mL syringe, and the infested area is wrapped with degreasing cotton then and placed into the soil. Eighteen plants were used per treatment. After two weeks, the degreasing cotton was removed. Over time, the plants continued to grow. To prevent growing roots from being exposed, they were buried with vermiculite. When hairy roots grew at the infected site and their length was more than 2 cm, the original root system below it was cut off to facilitate rapid growth, and the leaves on the shoots were removed to facilitate survival.

Transformation of A. Chinensis Leaves
The AcEXPA23 ORF was cloned into the pART-CAM-EGFP vector driven by the CaMV 35S promoter and the recombinant plasmid was subsequently transformed into A. chinensis leaves, according to the protocol outlined by Wang [73] (Table S2). Transgenic plants were obtained after approximately 6 months. Transformed plants were identified using PCR and qRT-PCR for the successful verification of transgene incorporation. Two transgenic kiwifruit lines with high AcEXPA23 mRNA expression were selected for morphological analysis.

Subcellular Localisation
The AcEXPA23 ORF with a mutated stop codon was cloned between the XbaI and SalI sites of the pBI221-GFP vector using T4 DNA ligase (Thermo Scientific, Waltham, MA, USA) ( Table S2). The recombinant and control plasmids were transformed into Arabidopsis leaf protoplasts as described previously [74]. After 18 h, GFP fluorescence was observed under a laser scanning confocal microscope (FV1000 viewer; Olympus, Tokyo, Japan) at 488 nm with argon-ion laser excitation and GFP was detected at 507 nm. Chloroplast autofluorescence was analysed using 488 nm argon-ion laser excitation, SP 630 nm IR detection, a pinhole of approximately 1.0 units, and an optical section thickness of approximately 0.5 µm.

Quantitative Real-Time Polymerase Chain Reaction
Primer Premier 5 software was used to design qRT-PCR primers for target genes (the primers used are listed in Table S2). The RNA extraction method and qRT-PCR were performed as described by Wu [39]. Actin (GenBank EF063572) was used as the normalised control gene [75] (Table S2). Three biological replicates were analysed. The relative expression was calculated using the 2 −∆∆Ct method [76].

Statistical Analysis
Excel 2010 (Microsoft Corporation, Redmond, WA, USA), IBM SPSS Statistics 25 (SPSS Inc., Chicago, IL, USA) and Origin 2021 (OriginLab Corporation, Northampton, MA, USA) were used for statistical analyses of the data. Differences between treatments were determined using ANOVA and mean comparisons were made using Fisher's least significant LSD.

Conclusions
We identified and analysed the AcExpansin gene family using bioinformatic methods. As such, 41 expansin genes were identified from kiwifruit and classified into four subfamilies, including AcEXPA, AcEXPB, AcEXLA, and AcEXLB. These genes were further analysed for physicochemical properties, chromosomal location, conserved domains, gene structure, and intraspecies collinearity. We found that 41 expansin genes were evolutionarily diverse and conserved at the DNA and protein levels. Finally, we used genetic transformation technology in the kiwifruit to demonstrate that overexpression of AcEXPA23 can promote the development of increased lateral roots in kiwifruit. This is of great significance for promoting the absorption of water and nutrients in kiwifruit to improve yield and quality.

Conflicts of Interest:
The authors declare no conflict of interest.